The present invention relates to the field of stable quinone- and photoreactive ketone phosphoramidite reagents designed for automated solid phase synthesis of oligomers terminating in a photoreactive moiety.
Attachment of a reporter group or another conjugation to oligonucleotides (ONs) has been the subject of considerable research as the resulting functionalised ONs display great potential as diagnostic or therapeutic agents (S. L. Beaucage, Comprehensive Natural Products Chemistry Vol. 7. Ed. E. T. Kool, Editors-in-Chief D. Barton and K. Nakanishi, Pergamon, 1999, 153-250). For example, ONs linked to anthraquinone (anthraquinone-ONs) and derivatives thereof have been prepared with the purpose of increasing the affinity towards complementary ONs via intercalation as well as for studies of site specific modification, cleavage, and crosslinking of duplex structures (K. Mori et al., FEBS lett. 1989, 249, 213-218; S. M. Gasper and G. B. Schuster, J. Am. Chem. Soc. 1997, 119, 12762-12771; L. G. Puskxc3xa1s et al., Nucleosides Nucleotides, 1995, 14, 967; H. Kang and S. E. Rokita; Nucleic acids Res., 1996, 24, 3896-3902). Another interesting application of anthraquinone-oligomers is the covalent immobilization of oligomers onto polymeric surfaces. Immobilisation of oligomers on various surfaces (Jacobsen, M. H. and Koch, T. WO 96/31557, 1996), such as plastic microtiter plates, microchips and micro particles has been achieved by various means and form the basis for a rapidly expanding technology within the field of diagnostic assays and disease screening assays (F. N. Rehman et al., Nucleic acids Res., 1999, 27, 649-655; P. W. Stevens et al., Nucleic acids Res., 1999, 27, 1719-1727; G. Ramsay, Nature Biotechnology, 1998, 16, 40-44).
Two general methods for covalent attachment of anthraquinone to oligomers by chemical means have previously been developed. The first method comprises coupling of an activated anthraquinone derivative with a pre-synthesized oligomer containing a reactive group such as a free primary amine function. This approach is illustrated by Kang and Rokita (Nucleic Acids Res., 1996, 24, 3896-3902) who synthesized 5xe2x80x2-end anthraquinone-oligodeoxynucleotides (ODNs) for the studies of site-specific and photo-induced alkylation of DNA. A dimethyl-anthraquinone-ODN conjugate was synthesized by coupling of the N-hydroxysuccinimide ester of 2-(3-propionic acid)-1,4-dimethylanthraquinone with 5xe2x80x2-amino hexamethylene linked ODN, obtained by standard automated solid phase synthesis. Anthraquinone-ONs have also been prepared by reaction of ONs containing xe2x80x9camino -linkerxe2x80x9d modified nucleobases or carbohydrate moieties with activated anthraquinone derivatives (Telser et al. J. Am. Chem. Soc. 1989, 111, 7226-7232; Akira et al. Bioconjugate Chem. 1993, 4, 499-508).
The other method comprises converting the anthraquinone into a synthon that can be used for automated solid phase synthesis, e.g. coupling of the anthraquinone to a phosphoramidite reagent. Depending on the availability of the building-block it can be argued that this direct incorporation is the most efficient approach, as the total synthesis of the anthraquinone-oligomers can be performed on an automated synthesizer.
Attachment of anthraquinone derivatives to ONs via direct incorporation has been approached by linking the anthraquinone group to the 2xe2x80x2-O position of a 5xe2x80x2-O-DMT (4,4xe2x80x2-dimethoxytrityl), 3xe2x80x2-O-phosphoramidite nucleoside reagents. K. Yamana et al. (Bioconjugate Chem. 1996, 7, 715-720) reported the synthesis of 5xe2x80x2-O-dimethoxytrityl 2xe2x80x2-O-(2-anthraquinonylmethyl)uridine 3xe2x80x2-O-cyanoethyl)-N,N-diisopropylphosphoramidite which was used for automated solid phase synthesis of anthraquinone-ONs.
De Mesmaeker et al. (Bioorganic, Medicinal Chem. 1997, 7, 1869-1874) described the synthesis of nucleoside dimers containing a 3xe2x80x2-5xe2x80x2 amide bond, wherein the nitrogen atom is attached to an anthraquinone molecule through a polymethylene linker. DMT-protection of the 5xe2x80x2-O position and phosphitylation of the 3xe2x80x2-O-position of the dimer afforded a reagent suitable for automated synthesis of anthraquinone-ONs.
A non-basic pseudonucleoside bearing an anthraquinone moiety has been prepared by K.-Y., Lin and M. Matteucci (Nucleic Acids Res. 1991, 19, 3111-3114, and U.S. Pat. No. 5,214,136). Starting from 2-chloro anthraquinone and diethanol amine an anthraquinone diol derivative was obtained which was converted into a DMT H-phosphonate reagent which was, subsequently, incorporated multiple times into an ODNs.
The above mentioned reagents allow incorporation of an anthraquinone functionality at different positions in an oligomer.
A few examples of phosphoramidite reagents not derived from nudeosides, developed exclusively for incorporation of anthraquinone at the 5xe2x80x2-terminus of an oligomer using automated solid phase synthesis have been reported.
K. Mori et al. (FEBS Lett. 1989, 249, 213-218) describe the synthesis of anti-HIV active 5xe2x80x2-linked anthraquinone-ODNs wherein an anthraquinone derivative is linked to an oligodeoxynucleotide (ODN) via either an ethylpiperazinyl or a hexamethylene linker. The 5xe2x80x2-linked anthraquinone-ODNs were obtained by coupling of a freshly prepared anthraquinone-ethylpiperazinyl phosphoramidite (obtained in 65% yield) or anthraquinone hexamethylene-linked phosphoramidite to the 5xe2x80x2-end of an ODN sequence using standard automated solid phase synthesis.
The anthraquinone-ethylpiperazinyl phosphoramidite reagent has also been described in WO 90/12802. The anthraquinone phosphoramidite was synthesised using the same procedure as described by K. Mori et al.: 1-chloroanthraquinone was reacted with 1-(2-hydroxyethyl)piperazine affording 1-(1-(2-hydroxyethyl)piperazinyl)anthraquinone which was phosphitylated by N,N-diisopropylphosphoramidochloride in the presence N,N-diisopropylethylamine to afford anthraquinone-ethylpiperazinyl phosphoramidite. The anthraquinone phosphoramidite was used without further purification in the automated solid phase synthesis of 5xe2x80x2-linked anthraquinone-ODNs used for attenuation or destruction of mammalian genetic expression or viral activity.
S. M. Gasperand G. B. Schuster (J. Am. Chem. Soc. 1997, 119, 12762-12771) described the synthesis of 5xe2x80x2-linked anthraquinone-ODNs with the purpose of establishing the fact that oxidative damage can migrate in double-stranded DNA. For this purpose, two anthraquinone phosphoramidites were synthesised: N-ethyl- and N-pentyl-2-anthraquinonecarboxamide phosphoramidite. The two phosphoramidites were synthesised from anthraquinone-2-carbonyl chloride, which was reacted with 2-amino-1-ethanol or 5-amino-pentanol to afford N-(2-hydroxyethyl)- and N-(5-hydroxypentyl)-2-anthraquinone-carboxamide, respectively. Reaction of these carboxamides with N,N-diisopropylmethyl-phosphonamides chloride afforded the corresponding phosphoramidites as thick dark red oils after column chromatography. Coupling of these anthraquinone phosphoramidites to the 5xe2x80x2-OH terminus of ODNs as the final step in a solid phase synthesis gave anthraquinone-ODN conjugates.
Large scale synthesis of anthraquinone-oligomer conjugates using automated solid phase chemistry requires readily available and relatively stable anthraquinone synthons. Initial attempts to synthesize stable anthraquinone phosphoramidite reagents revealed that the above-mentioned types of reagents appear to be unstable.
The synthesis of an anthraquinone phosphoramidite derivative of N-(6-hydroxyhexyl)-2-anthraquinone carboxamide using N,N,Nxe2x80x2,Nxe2x80x2-tetraisopropylphosphorodiamidite and tetrazole is described in Example 1. Attempted isolation of this cyanoethyl phosphoramidite led to decomposition. Use of the crude product, after filtration of the reaction mixture, directly onto the DNA synthesizer within one day also led to decomposition. Following, attempts to prepare a cyanoethyl phosphoramidite analog of the N-(2-hydroxyethyl) anthraquinonecarboxamide by reaction of N-(2-hydroxyethyl)anthraquinonecarboxamide with 2-cyanoethyl N,N-diisopropylphosphoramidochloridite in the presence of ethyldiisopropyl amine (see Example 2) or by the same procedure as described in Example 1 afforded, initially, a bright yellow foam after flash chromatography. Drying of this material under high vacuum over night resulted in a dark brown syrup, indicating decomposition. The fact that all of the above anthraquinone phosphoramidite reagents have to be used immediately after preparation makes them less suitable for synthesis of a large-scale synthesis of anthraquinone-oligomer conjugates.
The present invention relates to a stable phosphoramidite reagent, designed for automated solid phase synthesis of oligomers, of the general formula I 
wherein Y and Yxe2x80x2 each independently are selected from optionally substituted C1-6-alkyl or Y and Yxe2x80x2 together with the nitrogen to which they are bonded form a non-aromatic N-heterocyclic ring; W is selected from O and S; X is selected from optionally substituted C1-6alkyl and optionally substituted benzyl; RN is selected from hydrogen C1-4alkyl, optionally substituted benzyl, optionally substituted quinones, and nucleosides; and Q is selected from optionally substituted quinones, and optionally substituted photoreactive ketones, such as optionally substituted benzophenone.
The invention also relates to an oligomer comprising the following fragment: 
wherein Q and RN are as defined above for formula (I); W and Wxe2x80x2 are independently selected from O and S; and V is selected from optionally substituted C1-6-alkyl, optionally substituted benzyl, hydrogen, Li+, K+, Na+, and NH4+.
The present invention furthermore relates to a stable phosphoramidite reagent of the general formula II 
wherein Y and Yxe2x80x2 each independently are selected from optionally substituted C1-6-alkyl or Y and Yxe2x80x2 together with the nitrogen to which they are bonded form a non-aromatic N-heterocyclic ring; X is selected from optionally substituted C1-6-alkyl and optionally substituted benzyl; W is selected from O and S; Q is selected from optionally substituted quinones and optionally substituted photoreactive ketones; n is an integer from 1 to 10; and m is 0 or 1.
The invention also relates to an oligomer comprising the following fragment: 
wherein Q, n and m are as defined above for formula (II); W and Wxe2x80x2 are independently selected from O and S; and V is selected from optionally substituted C1-6-alkyl, optionally substituted benzyl, hydrogen, Li+, K+, Na+, and NH4+.
The applicant has successfully approached covalent coupling of synthetic oligomers onto carbon-containing polymers in two different ways. In the first approach, a photoprobe, consisting of an anthraquinone or benzophenone molecule linked to an electrophilic reactive group via an ethylene glycol linker, was coupled to a polymer surface by short time exposure to UV light. Subsequently, reaction between the electrophilic groups attached to the polymer and nucleophilic aminoalkyl ONs lead to immobilization of the oligomers.
The second approach involved automated solid phase synthesis of anthraquinone-oligomers or benzophenone-oligomers. Irradiation of an aqueous solution containing either the anthraquinone-oligomers or benzophenone-oligomers with soft UV light resulted in attachment of the anthraquinone-oligomers and benzophenone-oligomers to the polymer surface through a covalent bond between the anthraquinone moiety or benzophenone moiety and the surface to which the solution has been applied.
The present invention describes the synthesis of surprisingly stable quinone- and photoreactive ketone phosphoramidite reagents which do not suffer from the drawbacks described above. These new reagents are easily synthesised from commercially available starting materials. Contrary to previous described 5xe2x80x2-end anthraquinone labelling phosphoramidites, the phosphoramidite reagents according to the present invention are isolated as stable solid materials, which can be stored for several months at xe2x88x9220xc2x0 C. without loss of reactivity and incorporated in an oligomer, using standard automated solid phase synthesis. Similarly, benzophenone phosphoramidites according to the present invention are isolated as stable oils, which can be stored for several months at xe2x88x9220xc2x0 C. without loss of reactivity and incorporated in an oligomer, using standard automated solid phase synthesis.
As mentioned above, the present invention i.a. relates to a stable phosphoramidite reagent of the general formula I 
wherein Y and Yxe2x80x2 each independently may designate an optionally substituted C1-6-alkyl or Y and Yxe2x80x2 together with the nitrogen to which they are bonded form a non-aromatic N-heterocyclic ring.
Among the possible Y and Yxe2x80x2, the situation where Y and Yxe2x80x2 each designate ethyl or isopropyl, or together designate pyrrolidino, piperidino or morpholino seem especially interesting, and the situation where Y and Yxe2x80x2 both are isopropyl appears to be particularly interesting.
The substituent X is selected from the group consisting of optionally substituted C1-6-alkyl and benzyl. Examples of optionally substituted C1-6-alkyl are methyl, 2-cyanoethyl, 2-(4-nitrophenyl)ethyl, 2-(2-pyridyl)ethyl, 2-(4-pyridyl)ethyl, and 2-(C1-6-alkylsulfonyl)ethyl among which 2-cyanoethyl presently is the most preferred.
W is selected from O and S where O is most preferred.
RN is selected from hydrogen and C1-4-alkyl, such as methyl, ethyl, and isopropyl, optionally substituted benzyl, optionally substituted quinones attached via suitable linkers, e.g. methylene and polymethylene, and nucleosides attached via 5xe2x80x2-C through a methylene or polymethylene linker; preferably RN designates hydrogen.
Q represents a group selected from optionally substituted quinones and optionally substituted photoreactive ketones.
By the term xe2x80x9cquinonexe2x80x9d is understood a dihydroaromatic system wherein the xe2x80x94CH2xe2x80x94 groups are replaced by xe2x80x94C(xe2x95x90O)xe2x80x94. In the present context xe2x80x9cquinonexe2x80x9d covers quinones derived from di- or tetrahydroaromatic systems comprised by 1 to 5 fused carbon cyclic rings. illustrative examples of such quinones are derived from 1,4benzoquinone, 1,2-benzoquinone, naphtoquinone, anthraquinone, phenanthrenequinone, alizarin, rubiadin, lucidin, damnacanthal, munjistin, chrysophanol, frangula-emodin, aloe-emodin, morindone, and copareolatin. As mentioned above, quinones may be optionally substituted, however, it is presently believed that unsubstituted quinones, in particular unsubstituted anthraquinone and phenanthrenequinone, are especially preferred.
Examples of particular interesting photoreactive ketones are acetophenone, benzophenone, anthrone and anthrone-like heterocycles, i.e. anthrone wherein the group in 10-position is replaced by O, S, or NH. The photoreactive ketones can be optionally asubstituted as described below. Particular interesting photoreactive ketones are benzophenone and acetophenone of which unsubstituted benzophenone is presently most preferred.
In a preferred embodiment of the present invention, the phosphoramidite has the following structure: 
In a preferred embodiment of the present invention, the phosphoramidite has the following structure: 
When coupled to oligomers, e.g. ONs or ODNs, the reagents of the present invention lead to a novel class of oligomers. Thus, the invention furthermore relates to an oligomer comprising the following fragment: 
wherein Q and RN are as defined above for formula (I), W and Wxe2x80x2 are independently selected from O and S, V is selected from optionally substituted C1-6-alkyl, optionally substituted benzyl, hydrogen, Li+, K+, Na+, and NH4+ and xe2x80x9coligomerxe2x80x9d has the meaning defined below. In a preferred embodiment, Q represents anthraquinone, RN represent hydrogen, W and Wxe2x80x2 both represent O, and V is hydrogen.
The invention also relates to a phosphoramidite reagent of the formula II 
wherein Y and Yxe2x80x2 each independently may designate an optionally substituted C1-6alkyl or Y and Yxe2x80x2 together with the nitrogen to which they are bonded form a non-aromatic N-heterocyclic ring.
Among the possible Y and Yxe2x80x2, the situation where Y and Yxe2x80x2 each designate ethyl or isopropyl, or together designate pyrrolidino, piperidino or morpholino seem especially interesting, and the situation where Y and Yxe2x80x2 both are isopropyl appears to be particularly interesting.
The substituent X is selected from the group consisting of optionally substituted C1-6-alkyl and benzyl. Examples of optionally substituted C1-6-alkyl are methyl, 2-cyanoethyl, 2-(4-nitrophenyl)ethyl, 2-(2-pyridyl)ethyl, 2-(4-pyridyl)ethyl, and 2-(C1-6-alkylsulfonyl)ethyl among which 2-cyanoethyl presently is the most preferred.
W is selected from O and S where O is most preferred.
Q represent a group selected from optionally substituted quinones and optionally substituted photoreactive ketones. Illustrative examples of such quinones are derived from phenanthrenequinone, 1,4-benzoquinone, 1,2-benzoquinone, naphtoquinone, anthraquinone, alizarin, rubiadin, lucidin, damnacanthal, munjistin, chrysophanol, frangula-emodin, aloe-emodin, morindone, and copareolatin. As mentioned above, quinones may be optionally substituted, however, it is presently believed that unsubstituted quinones, in particular unsubstituted anthraquinone and phenanthrenequinone, are especially preferred.
Examples of particular interesting optionally substituted photoreactive ketones are benzophenone, amino-, hydroxyl-, halogen-, acyl-, nitro-, and cyanobenzophenone, of which unsubstituted benzophenone is presently most preferred.
n is an integer from 1 to 10. It is presently believed that variants where n is ranging from 1 to 4, such as 1, 2, 3 or 4, are particularly relevant.
m is 0 or 1.
In a preferred embodiment, Y and Yxe2x80x2 both are isopropyl and X designates 2-cyanoethyl
Coupling of phosphoramidite reagents of the general formula II to the termini of an oligomer affords oligomers containing the following fragment. Thus, the invention also relates to an oligomer comprising this fragment: 
wherein Q, n and m are as defined above for formula (II), W and Wxe2x80x2 are independently selected from O and S, and V is selected from optionally substituted C1-6-alkyl, optionally substituted benzyl, hydrogen, Li+, K+, Na+, and NH4+ and xe2x80x9coligomerxe2x80x9d has the meaning defined below. In a preferred embodiment, Q represents anthraquinone or phenanthrenequinone, W and Wxe2x80x2 both represent O, V is hydrogen, n is 1, and m is 0.
It should also be understood that the phosphoramidite reagents of the general formulas I and II can be coupled to the 3xe2x80x2-OH termini of an oligomer synthesized from 5xe2x80x2xe2x86x923xe2x80x2.
Preparation of Phosphoramidite Reagents
In a preferred embodiment, anthraquinone phosphoramidites were synthesised by the following procedures:
Synthesis of the anthraquinone phosphoramidite 3 is illustrated in FIG. 1 and was performed in two steps starting from commercially available anthraquinone-2-carboxylic add (1). Coupling of compound 1 with 3amino-1-propanol in the presence of benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexaflourophosphate (BOP) yielded the amide 2. Subsequently, phosphitylation of 2 using 2-cyanoethyl-N,N-diisopropylphosphoramido-chloridite afforded the anthraquinone phosphoramidite 3 as a red oil after aqueous workup. Redissolution of the crude product 3 in a minimum amount of anhydrous methylenechloride and subsequent precipitation in vigorously stirred petroleum ether at 0xc2x0 C. afforded 3 as a bright yellow powder. The product 3 was dried overnight at high vacuum and stored under nitrogen at xe2x88x9220xc2x0 C.
Synthesis of the anthraquinone phosphoramidite 5 is illustrated in FIG. 1 and was performed in one step starting from commercial available 2-(hydroxymethyl)anthraquinone (4). Phosphitylation of 2-(hydroxymethyl)anthraquinone (4) using the same procedure as described for the preparation of 3 afforded the corresponding phosphoramidite 5 as a yellow oil, which was coevaporated with anhydrous acetonitrile to afford 5 as a yellow solid material.
Alternatively, reaction of 2-(hydroxymethyl)anthraquinone (4) with 2-cyanoethyl-N,N,Nxe2x80x2,Nxe2x80x2-tetraisopropylphosphorodiamidite and tetrazole afforded the phosphoramidite 5 as a bright yellow solid material after filtration and aqueous workup.
Phosphoramidite 3 has been used in automated solid phase synthesis for a large number of anthraquinone-ODN conjugates. The phosphoramidite 3 was coupled directly to the 5xe2x80x2-OH termini of an ODN or via a 5xe2x80x2-hexaethyloxyglycol spacer (Spacer(trademark)) to an ODN as the final step in an automated solid phase synthesis on a Gene Assembler Special(copyright) synthesiser using a 0.1 M solution and a 5 min. coupling time. The coupling efficiency was estimated to be  greater than 98% as attempted coupling of another thymidine nucleoside (T) residue to a test sequence 5xe2x80x2-anthraquinone-T-3xe2x80x2 failed completely (no 4,4xe2x80x2-dimethoxytrityl-release was monitored). The two general types of anthraquinone oligonucleotide synthesised are illustrated in FIG. 2.
In a preferred embodiment, optionally substituted photoreactive ketone phosphoramidites, such as benzophenone phosphoramidites, were synthesised by the following procedures:
Synthesis of the anthraquinone phosphoramidite 8 was performed in two steps starting from commercially available benzoylbenzoic acid (6). Coupling of compound 6 with 3-amino-1-propanol in the presence of benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexaflourophosphate (BOP) yielded the amide 7. Subsequently, phosphitylation of 7 using 2-cyanoethyl-N,N-diisopropylphosphoramido-chloridite afforded the benzophenone phosphoramidite 8 as a pale yellow oil. This oil was used without further purification and stored under nitrogen at xe2x88x9220xc2x0 C.
FIG. 3 illustrates the synthesis of a benzophenone-phosphoramidite reagent. Its application for the preparation of benzophenone-oligonucleotide conjugates was analogous to that outlined in FIG. 2 for anthraquinone oligonucleotide conjugates.
Phosphoramidite 8 has been used in automated solid phase synthesis for a large number of anthraquinone-ODN conjugates. The phosphoramidite 8 may be coupled directly to the 5xe2x80x2-OH termini of an ODN or via a 5xe2x80x2-hexaethyloxyglycol spacer (Spacer(trademark)) to an ODN as the final step in an automated solid phase synthesis on a Gene Assembler Special(copyright) synthesiser using a 0.2 M solution and a 15 min. coupling time.
DNA oligomers carrying a 5xe2x80x2anthraquinone or a 5xe2x80x2benzophenone can be covalently immobilised on a solid support by irradiation and the immobilised oligomers are efficient in the capture of a complementary DNA oligomer.
As shown in FIGS. 6 and 7, both the AQ oligomers and the BP oligomers yield a clearly concentration dependent signal. When using a non-complementary sequence, no signal could be detected. It is concluded that both anthraquinone and optionally substituted photoreactive ketone oligomers, such as AQ and BP oligomers, can be covalently attached to a solid surface by irradiation and that oligomers attached in this way are able to hybridise to their complementary target DNA oligomers.
Definitions
In the present context, the term xe2x80x9cC1-6alkylxe2x80x9d means a linear, cyclic or branched hydrocarbon group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, pentyl, cyclopentyl, hexyl, cyclohexyl, preferred examples of xe2x80x9cC1-6-alkylxe2x80x9d are ethyl, propyl, iso-propyl, butyl, tert-butyl, isobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, in particular ethyl. Analogously, the term xe2x80x9cC1-4-alkylxe2x80x9d means a linear, cyclic or branched hydrocarbon group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, and tert-butyl.
In the present context, i.e. in connection with the terms xe2x80x9calkylxe2x80x9d, xe2x80x9cquinonexe2x80x9d and xe2x80x9cphotoreactive ketonesxe2x80x9d, the term xe2x80x9coptionally substitutedxe2x80x9d means that the group in question may be substituted one or several times, preferably 1-4 times, with group(s) selected from hydroxyl, amino, halogen, acyl, nitro and cyano, C1-6-alkoxy, C1-6-alkyl (only relevant for quinone and photoreactive ketones), formyl, carboxyl, C1-6-alkoxycarbonyl, C1-6-alkylcarbonyl, aryl, aryloxycarbonyl, arylcarbonyl, heteroaryl, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)aminocarbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkylcarbonylamino, carbamido, where C1-6-alkyl, aryl and heteroaryl may be substituted 1-5 times, preferably 1-3 times, with hydroxyl, acyl, C1-4-alkyl, C1-4-alkoxy, nitro, cyano, amino or halogen.
xe2x80x9cHalogenxe2x80x9d includes fluoro, chloro, bromo, and iodo.
In the present context, the term xe2x80x9coligomer(s)xe2x80x9d means oligonucleotides (ONs), oligodeoxynucleotides (ODNs), and derivatives thereof, such as ONs/ODNs modified in the carbohydrate moiety, e.g. Locked Nucleoside Analogues (LNAs), ONs/ODNs modified in the phosphodiester linkaged, e.g. phosphorothioates, phosphoramidates, and methylphosphonates, ONs/ODNs modified in the heterocyclic base, and xe2x80x9cbackbonexe2x80x9d modified ONs/ODNs, e.g. Peptide Nucleic Acids (PNAs). The oligomers may be from 1-1000 units, e.g. 1-1000 nucleotides, preferably 1-200, even more preferably 5-30 units, and each oligomer may comprise different classes of units, e.g. ODN-LNA conjugate. It should also be understood that the term xe2x80x9coligomerxe2x80x9d means oligomers synthesized from 3xe2x80x2xe2x86x925, terminating in a 5xe2x80x2-OH, as well as oligomers synthesized from 5xe2x80x2xe2x86x923xe2x80x2, terminating in a 3xe2x80x2-OH.